G-proteins are heterotrimeric guanine nucleotide binding proteins that regulate multiple cellular pathways in response to hormones. These proteins form a multigene family that possess similar modes of activation related to their overall structural homology. The hormone receptors that activate G-proteins are also structurally homologous and appear to regulate the activity of G-proteins by similar mechanisms. The discovery of oncogenes such as ras, also a GTP-binding protein with many similarities to normal G-proteins, and mas, actually the angiotensin receptor, suggests that G-proteins and their receptors are intimately involved in cellular growth control as well as in the regulation f cAMP levels, ion channels and PI turnover, itself a regulator of multiple cellular pathways. Rhodopsin, the photoreceptor of the mammalian rod cell regulates the activity of a cGMP phosphodiesterase via Gt, the rod cell G-protein. This light- activated process is directly analogous to hormone-mediated activation of other G-protein-coupled receptors. This proposal describes the modification of the bovine opsin cDNA by site-directed mutagenesis in order to define domains of the G-protein-coupled receptor that are responsible for the binding and activation of G-proteins. In addition mutational analysis will be performed to define sites of interaction with rhodopsin kinase, a rod cell protein that phosphorylates photolyzed rhodopsin reducing its ability to activate Gt. The role of arrestin in promoting desensitization of photolyzed, phosphorylated rhodopsin will also be examined. The rhodopsin phototransduction system serves as an excellent model system for studying the regulation of activity of G-protein-coupled receptors because of the ease of purification of large quantities of all of the proteins involved in this process. In addition, Gt behaves as an extrinsic membrane protein. Therefore kinetic studies can easily be performed to measure the binding of this protein to the rod cell photoreceptor and its subsequent activation.